Chemical Biology of Tea Catechins Tsutomu NAKAYAMA Laboratory of - PowerPoint PPT Presentation

Workshop Argentina-Japan “Bioscience and Biotechnology for the Promotion of Agriculture and Food Production” August 4 th 2009 Chemical Biology of Tea Catechins Tsutomu NAKAYAMA Laboratory of Molecular Food Engineering and Global COE Program, School of Food and Nutritional Sciences, University of Shizuoka

What are tea catechins ? Tea catechins are present in green tea and black tea. EC 6%, EGC 19%, ECg 14%, EGCg 59% In green tea. Antioxidant activity Antibacterial effect Epicatechin [EC] Epigallocatechin [EGC] Antimutagenicity Antihypercholesterolemia etc… B The four analogues show substantially different activities in assays in vitro. A C Inactivation of influenza virus ECg > EGCg >> EGC galloyl Antibacterial effect EGCg > ECg > EGC > EC Epicatechin gallate Epigallocatechin gallate [ECg] [EGCg]

Interaction between catechins and proteins (albumin) Part １

1- -1. Detection of EGCg 1. Detection of EGCg- -binding proteins with binding proteins with 1 redox cycling staining redox cycling staining OH nitroblue tetrazolium nitroblue tetrazolium OH electrophoresis (NBT) (NBT) HO O OH O ROS ROS OH OH O PVDF membrane PVDF membrane OH OH ROS (superoxide) ROS (superoxide) OH O PVDF membrane PVDF membrane OH O R nitroblue formazan nitroblue formazan Glycine, alkaline pH Glycine, alkaline pH

Conclusions obtained by the redox cycling staining 1. Tea catechins bind covalently human serum albumin, but only under oxidative conditions. 2. Under these conditions, human serum albumin is also oxidized. 3. A LC-MS study conducted in our laboratory revealed that the covalent binding occurred between EGCg and cystein of human serum albumin. Mol. Nutr. Food Res., 53, 709-715, 2009 Albumin stabilizes (-)-epigallocatechin gallate in human serum: Binding capacity and antioxidant property

1-2. Dynamic analysis of non-covalent binding of catechins to proteins by Quartz-Crystal Microbalance (QCM) Catechins injection HSA Binding amount QCM cells Gold AT-cut electrode Quartz Time (min) 1 / τ ＝ K on ［ Y ］＋ K off 1 / τ K on Host Guest Complex + X Y XY K off [XY] K a = = K on / K off [X] [Y] Conc. of catechins

Binding constants of catechins to human serum albumin (HSA) K on [M -1 S -1 ] K off [S -1 ] K a [M -1 ] Catechins 5.9 × 10 0 2.8 × 10 -3 5.8 × 10 3 EC 8.5 × 10 0 3.0 × 10 -3 3.4 × 10 3 EGC 5.0 × 10 2 3.6 × 10 -3 4.3 × 10 5 ECg 2.2 × 10 2 8.9 × 10 -4 4.3 × 10 5 EGCg OH OH OH OH OH OH OH OH O O HO HO O O HO HO OH OH OH OH O O OH OH OH OH OH OH O O OH OH OH OH EC EGC ECg EGCg

1-3. Affinity of catechins to HSA analyzed by HPLC with HSA column O Si N HSA NH H O Si HSA N NH H 3-aminopropyl silica-gel HPLC condition K HSA ＝ ( t R － t 0 ) / t 0 Column: SUMICHIRAL HSA (4.0 × 150 mm) Mobile phase: 20 ％ acetonitrile in 0.1 M t R ： retention time of a catechin (min) sodium phosphate buffer pH 5.0 t 0 ： retention time of unretained Flow rate: 0.9 mL/min molecules ( i.e. , citric acid) (min) Injection volume: 10 μ L UV detection: 200 nm

HPLC chromatogram of catechins with HSA column K HSA 100 2.89 EC 0.65 0 100 3.12 EGC 0.78 0 100 16.90 ECg 8.65 0 100 20.82 EGCg 10.89 0 0 20 40 60 Retention time (min)

Summary of Part 1 Structural factors governing affinity of catechins for HSA Number of hydroxy group OH of the B-ring contributing OH hydrogen bonding O HO OH O OH OH O OH OH The presence of galloyl moiety producing hyrophobic Hydrophobic region region

Part 2 Interaction between catechins and phospholipids investigated by HPLC and NMR

How do tea catechins interact with lipid membranes? Tea catechins Phospholipids

2-1. Immobilized Artificial Membrane (IAM) column t R : The retention time of the compound t 0 : The time for unretained molecules ( i.e. , citric acid) HPLC chromatogram with an IAM column

Phospholipophilicity of tea catechins The K IAM values correlated well with the amounts incorporated into the liposomes and with the partition coefficient obtained from n -octanol/PBS system.

2-2. Solution NMR study B 0 DMPC DHPC isotropic bicelle Isotropic bicelle solutions were prepared by the following conditions: DMPC : DHPC = 1 : 2 tea catechins : DMPC = 0.24 : 1 final lipid concentration: 8% w/v (D 2 O)

1 H NMR spectra (ECg) B 1 H NMR (400 MHz, D 2 O) A C galloyl galloyl B-ring ECg (free) ECg + bicelles The B-ring and galloyl moiety interact with phospholipid membranes.

Comparison of 1 H chemical shift change values (bicelles) 0.04 0.02 G1b γ β C2 C3 0.00 G3 G2 (CH 2 ) (CH 3 ) –0.02 G1a [ppm] α –0.04 –0.06 –0.08 –0.10 –0.12

1 H spin-lattice relaxation times ( T 1 ) [sec] Inversion recovery method (PD: 40 s) 2 T 1 Catechins (free) 3 Catechins ※ 4a + bicelles ※ Overlapped ※ 4b 6/8 FAST SLOW 2 ′ B ring τ c 5 ′ 6 ′ galloyl Molecular motion of B-ring and galloyl moiety was restricted 2 ″ , 6 ″ in the presence bicelles. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 T 1 [sec]

Location of ECg in the model membrane clarified by solution NMR OH HO in aqueous solution O OH (H 3 C) 3 N O O O OH OH P O O O OH OH O O O catechin (ECg) O C 12 H 25 C 10 H 21 intermolecular NOE interacting with bicelles

Summary of Part 2 The molecular-level interactions of tea catechins with lipid bilayers have been clarified based on: HPLC IAM column Affinity of tea catechins for phospholipid membranes Biosci. Biotechnol. Biochem. 72, 3289–3292 (2008). Solution NMR Chemical shifts ( 1 H NMR) B-ring and galloyl moiety of ECg and EGCg, and phospholipids γ -H, are involved in the interaction. T 1 relaxation times ( 1 H NMR) NOE effects B-ring and galloyl moiety are closely located to γ -H of ( 1 H– 1 H, 1 H– 13 C) phospholipids J. Agric. Food Chem. 55, 9986–9992 (2007).

Conclusions 1. Affinity for proteins EGCg > ECg >> EGC > EC Presence of galloyl moiety is the most decisive factor. Presence of hydroxy moiety in the B ring increases the affinity. Catechins interact with proteins both by hydrophobic bonding and hydrogen bonding. 2. Affinity for phospholipids ECg > EGCg >> EC > EGC Presence of galloyl moiety is the most decisive factor. Presence of hydroxy moiety decreases the affinity. Catechins interact with phospholipids in the surface of membranes only by hydrophobic bonding. 3. These results should be useful to clarify the mechanisms of the functions of tea catechins.